Wang CH et al / Acta Pharmacol Sin 2004 Aug; 25 (8):1027-1030
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©2004, Acta Pharmacologica Sinica Chinese Pharmacological Society Shanghai Institute of Materia Medica Chinese Academy of Sciences http://www.ChinaPhar.com
Intrathecal cdk5 inhibitor, roscovitine, attenuates morphine antinociceptive tolerance in rats1 Cheng-haung WANG2, Tsung-hsing LEE2, Yi-jung TSAI3, Jong-kang LIU3, Yann-jang CHEN4, Lin-cheng YANG2, Cheng-yuan LU2,5 2 Department of Anesthesiology, Kaohsiung Chang-Gung Memorial Hospital, 3Department of Biological Sciences, “National” Sun Yat-sen University, 4Center for General Education, “National” Yang-Ming University, Taipei 833, Taiwan, China
KEY WORDS roscovitine; cyclin-dependent kinase 5; morphine ABSTRACT AIM: To investigate the effect of cyclin-dependent kinase 5 (Cdk5) inhibitor roscovitine on the morphine antinociceptive tolerance development in rats. METHODS: Tail-flick test as pain threshold measurement and intrathecal injection techniques were used. RESULTS: Intrathecal roscovitine infusion alone produced an antinociceptive effect. Tolerance was induced by continuous intrathecal infusion of morphine 5 µg/h for 5 d. Coadministration of intrathecal roscovitine 1 µg/h for 5 d enhanced the morphine antinociceptive effect in tolerant rats. It also caused a shift in the morphine antinociceptive dose-response curve to the left when co-administered with morphine during tolerance induction, and caused a 67 % reduction in the increase in the ED50 of morphine (dose producing 50 % of the maximum response). CONCLUSION: Cdk5 modulation is involved in the antinociceptive tolerance of morphine. Intrathecal roscovitine administration could attenuate this tolerance development.
INTRODUCTION The most efficacious drugs used to relieve pain are the opioid analgesics. However, chronic administration of these drugs leads to the development of tolerance and dependence, processes that were intimately related to opioid addiction. Tolerance is manifested as a decreased potency of the drug, so that progressively larger doses must be administered to achieve a given level of analgesia. The processes underlying opioid tolerance involve complex compensatory changes in many opioid and nonopioid neuronal circuits[1,2]. The cellular
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Project supported by Kaohsiung Chang-Gung Memorial Hospital Research Plan (CMRP-1136),Taiwan, China. 5 Correspondence to Dr Cheng-yuan LU. Phn 886-7-717-2565. E-mail
[email protected] Received 2003-08-19 Accepted 2004-02-04
mechanism underlying the development of morphine tolerance remains controversial. It has been postulated that morphine tolerance is a result of (i) the up- or downregulation of opioid receptors (µ, δ, or κ subtypes), (ii) the uncoupling or desensitization of G-proteins, (iii) altered intracellular signaling, including via adenyl cyclase, protein kinase C, or nitric oxide, and (iv) the involvement of post-receptor neural events, particularly those involving the N-methyl-D-aspartate (NMDA) receptor, γ-aminobutyric acid, and monoamine neurotransmitter[3]. Chronic morphine exposure up-regulates the transcription factors cAMP-response-element-binding protein (CREB) and deltaFosB, both of which appear to mediate an aspect of tolerance[4,5]. DeltaFosB is thought to be a sustained molecular switch for addiction[4]. Cyclindependent kinase 5 (Cdk5) was shown to be a downstream target gene of delta FosB, and to regulate the
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Wang CH et al / Acta Pharmacol Sin 2004 Aug; 25 (8):1027-1030
effects of chronic cocaine exposure in mice[6]. Cdk5 is a member of the Cdk family of serine/threonine kinases and is so named because of its sequence homology to other Cdks[7,8]. It is activated by neuron-specific p35 proteins and exists as a large, multimeric complex associated with cytoskeletal proteins in the neurons, where it phosphorylates a wide variety of proteins, all of which have serine/threonine sites in (K/RT/SPXK)-type motifs[9]. Cdk5 and p35 are expressed predominantly in post-mitotic neurons, with essential roles in neuronal migration, neurite outgrowth, and the laminar configuration of the cerebral cortex[10-12]. Cdk5 has been shown to regulate NMDA receptor phosphorylation. Interestingly, Cdk5 is modulated by metabotropic glutamate receptors in neostriatal neurons[13]. Besides, opioid addiction was associated with hyperphosphorylation of neurofilament. Neuronal Cdk5 and its neuron-specific activator p35 play a major role in regulating the cytoskeleton dynamics[14]. These facts together imply a strong interaction between Cdk5 and morphine tolerance, which may be involved in the pain signal transduction. Roscovitine is a potent and selective inhibitor of Cdk5, and competes for the ATP-binding site of the kinase. ED50 for Cdk5 is 0.16 µmol/L[15]. Roscovitine 5 µmol/L significantly inhibits NMDA-induced long-term potentiation in hippocampal CA1 neurons[16]. In the present study, the contribution of Cdk5 modulation to morphine antinociceptive tolerance was evaluated in tolerant rats. METHODS AND MATERIALS Animals and groups Male Sprague-Dawley rats (Academia Sinica, Taiwan, China), weighing 280-320 g, were kept two per cage for at least 5 d after their arrival. The rats housed in a room with a 12:12 h darklight cycle, and a temperature of 22±0.5 ºC, with food (Lab Diet 5010; PMI Nutrition, Brentwood, MO, USA) and water ad libitum. The ethical guidelines specified by Chang-Gung Animal Ethics were followed throughout the study. Chronic intrathecal catheters were implanted under isoflurane anesthesia. Briefly, a polyethylene (PE-5) catheter, filled with 0.9 % saline, was advanced 8.5 cm caudally through an incision in the atlantooccipital membrane, and its tip positioned at the level of the lumbar enlargement. The rostral tip of the catheter was passed subcutaneously, and connected to an Alzet osmotic pump (200 µL capacity, 1 µL/h pump rate; Model 2001, Alza Corporation, Mountain View, CA, USA). Pump function was confirmed by weighing the pump before placement and after the experiment. Rats
showing neurological deficits after implantation were excluded. Rats were randomly divided into three groups. Group I received continuous intrathecal roscovitine 1 µg/h infusion for 5 d. Group II received continuous intrathecal morphine (5 µg/h) infusion for 5 d. Group III received both continuous intrathecal roscovitine 1 µg/h and morphine 5 µg/h infusions for 5 d. Each group consisted of 10 rats. Dosage was chosen due to preliminary result. Behavioral testing Tolerance to the antinociceptive effect of morphine was induced by continuous intrathecal infusion of morphine (5 µg/h) for 5 d. To investigate the effects of the Cdk5 inhibitor, roscovitine, on morphine tolerance, we calculate the maximum possible effect (MPE) for morphine antinociception after morphine tolerance was developed. The effects of roscovitine on the morphine antinociceptive dose-response curve were examined on the fifth day of tolerance induction. Nociceptive responsiveness was determined using the warm water (52 ºC) immersion tailflick test. Latency to the first rapid tail flick represented the behavioral end-point and cut-off latency is 10s to avoid tissue damage. The tail-flick test was performed daily. After a 5-d infusion, the tail-flick response was converted from a defined latency to the MPE after by morphine challenge (1, 10, and 20 µg, intrathecal infusion) as follows: MPE (%)=(maximum latency–baseline latency)× 100/cut-off latency (10 s)–C baseline latency. Drugs Roscovitine (LC Laboratories, Woburn, MA, USA) was dissolved in dimethyl sulfoxide (Me2SO), which has no significant effect on antinociception[17]. Statistical analysis Morphine antinociceptive dose-response latency was analyzed by computer-assisted linear regression (Cricket Graph 1.32; Islandia, NY, USA). The ED50 was defined as the morphine dose that inhibited 50 % MPE measured by the tail-flick test and was calculated using the linear regression equations. All data were presented as mean±SEM and subjected to analysis of variance and the Dunnett test, and P
